Advanced search
1 file | 772.02 KB Add to list

Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum)

Author
Organization
Abstract
Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof.

Downloads

  • De Bruyn et al. 2020 Frontiers in Genome Editing 2 604876.pdf
    • full text (Published version)
    • |
    • open access
    • |
    • PDF
    • |
    • 772.02 KB

Citation

Please use this url to cite or link to this publication:

MLA
De Bruyn, Charlotte, et al. “Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium Intybus Var. Foliosum).” FRONTIERS IN GENOME EDITING, vol. 2, 2020, doi:10.3389/fgeed.2020.604876.
APA
De Bruyn, C., Ruttink, T., Eeckhaut, T., Jacobs, T., De Keyser, E., Goossens, A., & Van Laere, K. (2020). Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum). FRONTIERS IN GENOME EDITING, 2. https://doi.org/10.3389/fgeed.2020.604876
Chicago author-date
De Bruyn, Charlotte, Tom Ruttink, Tom Eeckhaut, Thomas Jacobs, Ellen De Keyser, Alain Goossens, and Katrijn Van Laere. 2020. “Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium Intybus Var. Foliosum).” FRONTIERS IN GENOME EDITING 2. https://doi.org/10.3389/fgeed.2020.604876.
Chicago author-date (all authors)
De Bruyn, Charlotte, Tom Ruttink, Tom Eeckhaut, Thomas Jacobs, Ellen De Keyser, Alain Goossens, and Katrijn Van Laere. 2020. “Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium Intybus Var. Foliosum).” FRONTIERS IN GENOME EDITING 2. doi:10.3389/fgeed.2020.604876.
Vancouver
1.
De Bruyn C, Ruttink T, Eeckhaut T, Jacobs T, De Keyser E, Goossens A, et al. Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum). FRONTIERS IN GENOME EDITING. 2020;2.
IEEE
[1]
C. De Bruyn et al., “Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum),” FRONTIERS IN GENOME EDITING, vol. 2, 2020.
@article{8686444,
  abstract     = {Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof.},
  articleno    = {604876},
  author       = {De Bruyn, Charlotte and Ruttink, Tom and Eeckhaut, Tom and Jacobs, Thomas and De Keyser, Ellen and Goossens, Alain and Van Laere, Katrijn},
  issn         = {2673-3439},
  journal      = {FRONTIERS IN GENOME EDITING},
  language     = {eng},
  pages        = {12},
  title        = {Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum)},
  url          = {http://dx.doi.org/10.3389/fgeed.2020.604876},
  volume       = {2},
  year         = {2020},
}

Altmetric
View in Altmetric